AOAC ERP Gluten Assays - March 2019

and measure in a microtiter plate reader at 450 nm vs air within 30 min after stopping the reaction. Do not reuse wells of the plate. Use separate pipet tips for each standard and each sample extract to avoid cross-contamination. Use a multistepper pipet for adding the conjugate, substrate/ chromogen, and stop solution. Use a single tip for each of these components. Components and procedures of test kit have been standardized for use in this procedure. Do not interchange individual components between kits of different batches (lot numbers). Do not freeze any of the kit components. Carefully dilute the components that are included in the kit as concentrates; avoid contaminations by airborne cereal dust or dirty laboratory equipment. Wear gloves during preparation and performance of the assay. Clean surfaces, glass vials, mincers, and other equipment with 60% ethanol. Carry out sample preparation in a room isolated from ELISA procedure. Check for prolamin contaminations of reagents and equipment. F. Preparation of Test Samples Weigh 5 g sample and grind to a powder as fine as possible to obtain maximal surface. Weigh 0.25 g of the solid ground sample or use 0.25 mL of a liquid sample in a 10 mL glass vial and add 2.5 mL cocktail. Close vial and mix well (avoid cross-contamination). If tannin- and polyphenol-containing samples (e.g., chocolate, chestnut, or buckwheat) are prepared, add an additional 0.25 g skim milk powder (food quality) to the sample–cocktail solution. Incubate for 40 min at 50°C (122°F) in a water bath. Let sample cool down; then mix with 7.5 mL 80% ethanol. Close vial and shake for 1 h upside down or by a rotator at room temperature (20–25°C/68–77°F). Centrifuge 10 min at 2500 g at room temperature (20–25°C/68–77°F). Remove the supernatant (extract) in a screw-top vial and keep for testing. Dilute the sample at least 1:12.5 (1 + 11.5, 0.1 + 1.15 mL) with the prepared sample dilution buffer (depending on the expected prolamin content of the sample). Dilute serially from the first dilution, if necessary mixing thoroughly each time before diluting further. Use 100 µL per well in the assay. G. Preparation of Components Delivered with Kit ( a )  Sample diluent . — Provided as a concentrate (5-fold). Only the amount which is actually needed should be diluted 1:5 (1 + 4) with distilled water (e.g., 3 mL concentrate + 12 mL distilled water, sufficient for the dilution of 10 samples). Make sure that the buffer is not contaminated with gliadin. ( b )  Antibody enzyme conjugate.— (Bottle with red cap.) Provided as a concentrate (11-fold). Since the diluted enzyme conjugate solution has a limited stability, only the amount that is actually needed should be diluted. Before pipetting, the conjugate concentrate should be shaken carefully. For reconstitution, the conjugate concentrate is diluted 1:11 (1 + 10) with distilled water (e.g., 200 μL concentrate + 2.0 mL distilled water, sufficient for two microtiter strips). Take care that the water is not contaminated with gliadin. ( c )  Washing buffer .—Provided as a 10-fold concentrate. Before use, the buffer must be diluted 1:10 (1 + 9) with distilled water (i.e., 100 mL buffer concentrate + 900 mL distilled water). Prior to dilution, dissolve any crystals formed by incubating the buffer in a water bath at 37°C (99°F). The diluted buffer is stable at 2–8°C (35–46°F) for 4 weeks.

( 7 ) Show reproducible affinity, sensitivity, specificity, and stability from batch to batch for more than 1 year ( 8 ) Monoclonal antibodies are preferred; polyclonal antibodies can be used if they fulfill the same specificity criteria to react with wheat, rye, and barley to 100% and have no cross-reactivity to oat, maize, teff, and others D. Reagents Items ( a )–( i ) are available as a test kit (RIDASCREEN® Gliadin; R-Biopharm AG, Darmstadt, Germany). All reagents are stable for 18 months from date of manufacture at 2–8°C (36–46°F). Refer to kit label for current expiration. Equivalent antibodies may be used for ( a ) and ( c ) provided they satisfy characteristic criteria in C . ( a )  Antibody-coated microwell strips .—Monoclonal antibodies are coated in 20 mM phosphate buffered saline (PBS), pH 6.0, onto a set of twelve 8-microwell strips (NUNC, Roskilde, Denmark), containing 0.01% sodium azide as preservative. ( b )  Wash buffer concentrate .—100 mL/bottle, 10x concentrate. Contains a final concentration of 20 mM PBS (0.9% sodium chloride) with 0.1% Synperonic and 0.01% bronidox L as preservative. ( c )  Peroxidase-labeled antibody. —One vial (1.2 mL, 11x concentrated). ( d )  Gliadin ready-to-use standards (antigen). —Six vials (1.3mL each, ready to use). Prepared by Sigma gliadin or own preparation, dissolved in 60% ethanol at a concentration of 1 mg/mL. Solution is further diluted in 20 mM PBS–Tween (0.9% sodium chloride, 0.05% Tween 20) containing 0.22% fish gelatin (Sigma) to 0, 5, 10, 20, 40, and 80 ng/mL gliadin, calibrated to the Working Group on Prolamin Analysis and Toxicity (WGPAT) gliadin (86% highly purified gliadin from 40 different European wheat varieties). ( e )  Substrate .—One vial, 7 mL (urea peroxide). ( f )  Chromogen .—One vial, 7 mL (tetramethylbenzidine in methanol). Can be added either separately or mixed 1 + 1 with ( e ) before pipetting. ( g )  Stop solution .—One vial, 14 mL (1 N H 2 SO 4 ). ( h )  Sample dilution buffer .—60 mL, 5x concentrate. Contains a final concentration of 20 mM PBS–Tween (0.9% sodium chloride, 0.05% Tween 20) with 0.22% fish gelatin (Sigma) and 0.01% Kathon as preservative. ( i )  Cocktail solution .—One vial, 105 mL. Recommended but not provided with the test kit: ( a )  Skim milk powder.— Food quality. ( b )  Samples .—Three control samples (powder), one nongliadin- containing sample (rice flour) and two prolamine-contaminated maize samples (A and B, concentration given by a certificate), which can be extracted with 60% ethanol and diluted further with the sample dilution buffer to control the test from run to run. E. General Instructions Store kit at 2–8°C (35–46°F). Let all kit components come to 20–25°C (68–77°F) before use. Return any unused microwells to their original foil bag, reseal them together with the desiccant provided, and store at 2–8°C (35– 46°F). The colorless chromogen is light-sensitive; therefore avoid exposure to direct light. Include ready-to-use standards in duplicates to each run of diluted sample extracts in duplicates. Add the diluted antibody– POD conjugate (diluted by water) to all wells. Add substrate and chromogen simultaneously. Stop the reaction with stop solution,

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